Clone-based physical maps are central to the establishment of an accurate sequence of a complex genome and anchoring the sequence to a chromosomal location. Physical maps produced with large-insert bacterial artificial chromosome (BAC) libraries also provide the cloned DNA necessary for functional studies that are critical for the interpretation of the sequence of the human genome. In order to expand the number of genomes that are amenable to clone-based physical mapping, many new BAC libraries are currently being made from a wide range of vertebrates. Comparative genomics offers a powerful tool for both interpretation of the human genome and improving the efficiency and throughput of building physical maps. Specifically, comparative sequencing can identify functional elements of the human genome through the detection of evolutionarily conserved sequences. In addition, since genome organization is highly conserved among groups of related vertebrates, such as mammals, the map and sequence of the human genome can provide a valuable reference point for 're-mapping' other genomes through the use of orthologous markers. The purpose of the research proposed here is to apply comparative genomics toward the development of practical methods for constructing physical maps from a diverse range of vertebrates as a means of facilitating the use of new BAC libraries by the biomedical research community. To accomplish this, universal probes derived from evolutionarily conserved sequences will be designed and optimized for screening single or multiple vertebrate BAC libraries not associated with extensive sequence resources, and thus provide a basis for building physical maps in species that otherwise would be inaccessible to targeted physical mapping. The experience gained from this work will then be applied to the creation of a novel genome-wide database of universal probes to be made available to the public. The resulting information and technology will be applicable to the simultaneous construction and comparison of clone-based physical maps in multiple species at any genomic locus.